LAUSR

2,4-Diaminopyrimidine (trivially K) and imidazo[1,2-a]-1,3,5-triazine-2(8H)-4(3H)-dione (trivially X) form a nucleobase pair with Watson-Crick geometry as part of an artificially expanded genetic information system (AEGIS). Neither K nor X can form a Watson-Crick pair with any natural nucleobase. Further, neither K nor X has an accessible tautomeric form or a protonated/deprotonated state that can form a Watson-Crick pair with any natural nucleobase. In vitro experiments show how DNA polymerase I from E. coli manages replication of DNA templates with one K:X pair, but fails with templates containing two adjacent K:X pairs. In analogous in vivo experiments, E. coli lacking dKTP/dXTP cannot rescue chloramphenicol resistance from a plasmid containing two adjacent K:X pairs. These studies identify bacteria able to serve as selection environments for engineering cells that replicate AEGIS pairs that lack forms that are Watson-Crick complementary to any natural nucleobase.